Data from several laboratories indicate that bone contains and/or secretes several transforming and non-transforming growth activities, and Sm-C (IGF-1) like material. Since the development and maintenance of bone tissue is dependent both on osteoblast activity and the timely replacement of aged osteoblasts from the precursor pool, a role for these growth factors in bone cell proliferation and possibly differentiation is indicated. Our interest in bone cell differentiation has in the past led us to attempt separation of bone cells with different phenotypes. We examined the cellular source and target cells for these bone derived growth factors, and concluded that the osteoblastic cells released late during collagenase digestion of bone are a major source of growth activity when compared to early release less osteoblastic cells, producing at least two growth inducing factors of MW 24,000 and 11,000 daltons, and a material that appears to be immunologically related to Sm-C (IGF-1). We now plan to compare and purify the growth inducing activities present in osteoblasts bone tissue and medium conditioned by osteoblasts. We also propose to test their effects on proliferation and differentiation of three classes of bone cells that we have succeeded in isolating using bone matrix digestion with pure collagenase, followed by a collagenase protease mixture, to prepare early released fibroblastic cells, intermediate cells, and late released osteoblastic cells. In addition to the traditional osteoblastic characteristics (alkaline phosphatase, PTH responsiveness) we have demonstrated phenotypic differences among these three classes of bone cell using biochemical markers such as EGF receptor number and affinity and endogenous growth factor production. The latter two classes of cells have different numbers of high affinity EGF receptors. We plan to further separate these two cell types using flow cytometry to select cell groups on the basis of total EGF and fluorescent anti-EGF binding. We will study whether the biochemical characteristics of these cells permit classification as osteoblasts, intermediate osteoblasts and/or non-osteoblasts. Finally the ability of less-or-non-osteoblastic bone cells (obtained by the above procedures) to acquire the osteoblastic phenotype after various treatments and/or exposure to bone growth factors will be followed. The ultimate aims of these studies are 1) to isolate homogeneous bone cell populations in order to 2) study bone cell development and 3) the role of endogenous growth factors in this process.